摘要： 通过不同的分离方法和培养条件探索鲫鱼肝细胞的原代培养,以建立稳定的鲫鱼肝细胞原代培养模型。用台盼蓝染色检测细胞存活率;用WST-1检测细胞活力,同时观察长期培养过程中肝细胞的形态变化。结果显示:组织块培养4～5 d后肝细胞从组织块中迁出并增殖,但这种方法所需时间长且细胞不易收集;机械分散法收集的细胞少,细胞存活率低;而胰蛋白酶消化法获得良好稳定的分离和培养效果。用0.1%胰蛋白酶消化30 min,每克肝重可收集1.47×108个细胞,平均存活率为91.43%。培养基和血清浓度均会影响肝细胞的贴壁和生长。在含有10%(V/V)胎牛血清(FCS)M199培养基中,4%CO2、28℃,肝细胞培养8～9 d后传代。

关键词: 鲫鱼, 肝细胞, 胰蛋白酶, 原代培养

Abstract: In order to establish a stable primary culture model of hepatocytes in Carassius auratus,3 different isolation methods(tissue culture,mechanical speration and pancreatin digestion) and culture conditions of primary hepatocytes culture were compared in the present study.The survival rate was tested with trypan blue exclusion and the viability was assessed with WST-1;the morphological changes of cells in long-term culture were also observed.The results showed that in tissue culture,hepatocytes migrated from liver and proliferated after 4-5 days,compare with the other methods,it took much longer time for the hepatocytes to move out and it was difficult to collect the cells for further culture.The number and viability of the hepatocytes obtained by mechanical separation were obviously lower than by pancreatin digestion.The liver tissue digested by 0.1% trypsin for 30 min yielded 1.47×108 cell/g(liver weight) and the viability was 91.43%.Both culture medium and concentration of serum influenced the attachment and proliferation of the hepatocytes.The cell cultivated in M199 basal medium supplemented with 10% fetal calf serum at 28℃,4% CO2 for 8-9 days can sub-cultured

Key words: Carassius auratus, Hepatocytes, Trypsin, Primary culture

中图分类号: 

• S917